This laboratory will evaluate the mechanism of the membrane transport of methotrexate, folic acid, dihydrofolic acid, and the tetrahydrofolates in mammalian cells. The effect of methotrexate on the transport of the naturally-occurring folates will be assessed and distinguished from the effect that methotrexate might have on the metabolism of these compounds. The investigative approach will employ both analyses of transport fluxes in Ehrlich Ascites tumor cells, in vitro, as well as biochemical analyses of folate compounds accumulated within cells. Studies will be continued to further evaluate the role of intracellular methotrexate in excess of that required for complete association with high-affinity binding sites in the inhibition of RNA, protein as well as DNA synthesis by this agent. The mechanism by which vinca alkaloids inhibit the uphill transport of the model amino acid, gamma-aminoisobutyric acid will be further explored. Studies will evaluate if this effect is secondary to a primary vinca-induced alteration in the electrochemical-potential difference for sodium or potassium across the cell membrane. An investigative approach will be employed to evaluate whether this inhibitory effect is related to an interaction between vinca alkaloids and microtubules or an interaction with other cellular elements. The mechanism of the membrane transport of actinomycin D will be further characterized. The relationship between intracellular binding of this agent and inhibition of RNA synthesis will be evaluated.